PROJECT SUMMARY Whether through active or passive immunization, broadly neutralizing antibodies (bNAbs) will likely form a cornerstone of HIV prevention. This application proposes an ancillary study of the African component of the antibody-mediated prevention trial (AMP) (HVTN 703/HPTN 081). The AMP study is the first proof-of-concept trial to evaluate the safety and efficacy of a bNAb (VRC01) to block HIV acquisition. Our team is involved in the laboratory investigations to measure serum levels associated with protection, and through sieve analysis, to determine how predicted genotypic features correlate with prevention efficacy. The goal of this application is to provide a deeper understanding on how HIV-1 diversity affects the amount of antibody needed to prevent infection; and the clinical consequences of infection with VRC01-resistant viruses. The extreme diversity of circulating HIV strains, together with the flexibility of the Envelope protein (Env) in evading immune pressure, could threaten the effectiveness of immunization studies. Utilizing sequence data and functional env clones from AMP breakthrough infections, the first aim will provide an up-to-date bNAb sensitivity profile of circulating strains, providing data useful for future clinical trials. This analysis will be done using the standardized Env-pseudotyped assay, and a subset will also be evaluated using infectious env clones generated in PBMCs to verify these data. The parent protocol sieve analysis will predict genetic features that affect VRC01 neutralization susceptibility and resistance, and the second aim of this application will experimentally validate these predictions, as well as evaluate how the cognate Env of breakthrough infections influences the impact of these mutations. This will provide experimental data on how changes in envelope affect the amount of antibody needed for prevention of infection. Lastly, VRC01 escape mutations have been associated with reduced viral fitness and we will compare fitness of VRC01-resistant and sensitive viruses and determine how this affects key clinical markers such as HIV viral load and CD4 counts. The HVTN 703/HPTN 081 trial provides a unique opportunity to define the impact of diversity on bNAb efficacy in a sub-Saharan Africa setting where new prevention approaches are still urgently needed. This study will be led by investigators at the University of Cape Town and the Fred Hutchinson Cancer Research Center, in collaboration with the National Institute for Communicable Diseases, and includes a training partnership with North-West University in Mahikeng, South Africa. Information generated in this proposal will support the next phase of combination passive immunization trials and will provide seminal data to understand the prevention efficacy of VRC01.